We have demonstrated that compounds based on an S-acyl-2-mercaptobenzamide thioester (SAMT) template specifically eject zinc from the C-terminal zinc-binding domain of NCp7 via acyl transfer from the thioester to the sulfur of a zinc-coordinating cysteine residue. The same residues are modified in infected cells treated with SAMT compounds. The zinc-binding domains of NCp7 are excellent targets for the development of new antiretroviral and microbicidal agents because of their structural conservation and the broad range of functions of NCp7 in the viral replication cycle. The SAMT compounds are virucidal and inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems;a lead compound was also shown to inhibit viral transmission in 5/6 treated rhesus macaques when applied vaginally 30 min prior to virus exposure. As recent studies with topical microbicides have shown that patient compliance is a major impediment to efficacy, we evaluated the effect of combining a lead SAMT compound with a partially-active vaccine for reducing HIV infection. Using a non-human primate model of vaginal HIV-1 transmission, we examined whether the modest protective effect of a prime-boost T-cell-based vaccine would be enhanced when used in combination with the SAMT. We specifically used a sub-optimal dose of the SAMT to more rigorously test the combination and make any effect of the combination clearer. This dose had been previously shown not to have a significant effect on HIV infection in cervical explant assays, but was effective at reducing the initial viral infectivity and infectivity of progeny virus. Using this system, we found that a significant delay in SHIV acquisition (Log-rank test;p=0.0416) was seen only in vaccinated macaques that were repeatedly challenged in the presence of the topical microbicide. Additionally, peak acute viremia was lower (Mann-Whitney test;p=0.0387) and viral burden reduced (Mann-Whitney test;p=0.0252) in the combination-treated animals. These results suggest that the combination of a vaccine and topical microbicide is a promising means to prevent transmission of HIV. We are currently pursuing further studies to elucidate the mechanism of the enhanced protection and to determine the effect when using a higher dose of the SAMT compound in combination with an improved vaccine. While conducting our studies on the mechanism of viral inactivation in cells, we observed that the thiol generated by reaction of the SAMT with NCp7 can be recycled intracellularly to form an active thioester. This novel mechanism of action has allowed us to develop pro-drugs, as the thioesters are not stable at the low pH of vaginal fluid or at the neutral pH of plasma. We have designed a series of molecules in which we have systematically modified the pro-drug protecting group and evaluated the activity and stability of the resulting compounds. These efforts will help pinpoint lead pro-drug compounds for use topically and systemically that retain activity with improved stability. Overall, our research continues to demonstrate the viability of the SAMTs as candidate microbicides for the prevention of HIV transmission.